Anti-human spermine oxidase monoclonal antibody and application thereof
A technology of monoclonal antibody and oxidase, applied in anti-enzyme immunoglobulin, biochemical equipment and methods, instruments, etc., can solve problems such as limiting SMO research
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Embodiment 1
[0048] Embodiment 1, the preparation of the monoclonal antibody of anti-human spermine oxidase
[0049] 1. Preparation of SMO antigen
[0050] The pET-15b / hSMO plasmid (containing the full-length coding DNA sequence of human SMO, that is, the DNA sequence shown in the sequence table SEQ ID NO: 1, encoding the protein shown in SEQ ID NO: 2, constructed in the previous work of our research group) Escherichia coli BL21(DE3) was transformed, and the successfully transformed monoclonal strain was picked and transferred to LB medium containing ampicillin for culture. When the bacteria grew to the logarithmic phase, IPTG was added to a final concentration of 1 mmol / L, and cultured at 37 °C for 6 h. Take 200 L of the bacterial solution before and after IPTG induction, centrifuge to remove the supernatant, dissolve the bacterial pellet in 40 L of ddH2O, add 10 L of 5×SDS loading buffer, boil at 100 °C for 5 min, and perform 12% SDS -PAGE gel electrophoresis, Coomassie brilliant b...
Embodiment 2
[0065] Embodiment 2, the specificity analysis of monoclonal antibody
[0066] 1. Determination of antibody titer by ELISA
[0067] The purified SSAT protein was diluted with coating buffer to a final concentration of 1 mg / L, and this solution was used to coat a 96-well ELISA plate with 100 L per well. After overnight coating at 4°C, anti-SMO monoclonal antibody solution at a dilution of 1:2 000 to 1:512 000 was added and incubated at 37°C for 1 h, then HRP-labeled goat anti-mouse IgG (1:5 000) was added and incubated at 37°C for 1 hour. After h, the color was developed by the TMB method. The determination of the antibody titer is the same as that in the "Polyclonal Antibody Serum Titer Detection" section in Example 1.
[0068] 2. Western blot detection of antibody specificity
[0069] The following protein samples were used for Western blot analysis: BL21 (DE3) empty bacteria; BL21 (DE3 ) transformed with pET-15b / SMO; BL21 (DE3 ) transformed with pET-15b / SMO and induc...
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